Confocal microscope and method for confocal microscopy, with automatic focussing

Abstract

The invention relates to a confocal microscope (1) comprising: - an excitation light source (5) for an excitation light beam (6); - an excitation light beam splitter (7); - a point detector (14) having detector optics (13) for sample light (10) returning from a sample (2); - a microscope objective (4); - a movement device (3) for adjusting a relative distance (AB) between the microscope objective (4) and the sample (2); - an analysis device (16) comprising an analysis beam splitter (8) for coupling out a portion (11a) of the sample light (10) returning from the sample (2), analysis optics (12) for focussing the coupled-out portion (11a) towards an analysis detector (17), the analysis detector (17) for detecting the coupled-out portion (11a), and a modulation device (22) with which the refractive power of the analysis optics (12) and / or a relative distance (RA) between the analysis detector (17) and the analysis optics (12) can be varied cyclically over time according to a modulation signal (140), wherein a detector signal (150) from the analysis detector (17) is used to determine a focus time point (tx) at which the coupled-out portion (11a) is focussed on the analysis detector (17) and with which a distance control signal for the movement device (3) is generated. The confocal microscope enables the distance between the sample and the microscope objective to be automatically adjusted in a simple and reliable manner.

Description

[0001] experienced for confocal

[0002] The invention relates to a confocal microscope for imaging a sample, in particular the surface of the sample. The invention further relates to the use of such a confocal microscope and a method for imaging a sample using confocal microscopy.

[0003] In confocal microscopy, an excitation light beam is focused onto a sample, typically the surface, using a microscope objective to focus it at a point. Sample light emanating from the sample is imaged via the microscope objective and detector optics onto a zero-dimensional detector (also called a point detector). A so-called confocal pinhole (also called a detector aperture or input aperture of the point detector), which is located in a conjugate plane of the microscope objective, ensures that essentially only sample light from the illuminated point on the sample reaches the point detector. Stray light is not imaged onto the point detector or is blocked by the detector aperture. To image an extended area of ​​the sample, different locations within the sample are measured sequentially (scanning the sample).

[0004] In confocal microscopy, the excitation light is concentrated in a very small volume of the sample, allowing even comparatively weak interactions with the excitation light, such as fluorescence light generated in the sample or Raman scattered light, to be measured. On the other hand, there is a significantly higher resolution in the axial direction (direction of beam propagation) compared to a conventional light microscope. While this enables the measurement of samples in three dimensions, it also makes the investigation of uneven, rough, or tilted sample surfaces significantly more difficult. In the case of opaque samples, the intensity of the measured signal of the sample light decreases as soon as the focus of the excitation light no longer coincides with the sample surface.Finally, even with a flat sample, the axial distance of the sample from the microscope objective can change during scanning, for example due to temperature changes ("drift"), which also complicates the measurement of the surface. In other words, when scanning the sample, the sample or its surface risks becoming out of focus of the excitation light.

[0005] Various methods have become known in the prior art for tracking the distance between a sample and a microscope objective during scanning of an imaging area of ​​a sample.

[0006] In the internet article "Topography Confocal Raman Imaging using True Surface Microscopy," WITec Instruments Corp., Maryville, Tennessee, USA, dated September 1, 2011, and posted on Spectroscopy online (www.sectroscopyonline.com), it is proposed to first acquire a first scan of the topography of the desired imaging area of ​​the sample using a confocal chromatic sensor, and then to perform a Raman image using confocal microscopy in a second scan along the learned surface. This procedure is quite time-consuming due to the two scans required, and furthermore, it generally cannot compensate for drift in the confocal microscope.

[0007] The company publication "PureFocus 850 Manual Version 2.4" from Prior Scientific, Ltd., Cambridge, UK, dated June 2022, pages 6-8, describes an autofocus functionality for existing microscope systems for installation between the objective lens and the tube lens. It proposes directing an auxiliary laser beam at a specified angle onto the sample surface via an adjustable collimating lens, a dichroic mirror, and the objective lens. One half of the laser beam is shaded with a knife-edge diaphragm. Light from the auxiliary laser reflected from the sample is imaged via the objective lens and the collimating lens onto a spatially resolving detector. Depending on the distance of the sample from the imaging optics, the position of the line on the spatially resolving detector changes, providing information for automatically controlling the focus position of the microscope and allowing the sample to be maintained in focus.The disadvantage of this approach is, firstly, the structural complexity of the auxiliary laser, whose wavelength should not overlap with the wavelength of the light required for sample analysis or generated during sample analysis. Secondly, if the sample is tilted unfavorably, the reflected laser beam may be deflected so far that it is no longer imaged onto the spatially resolving detector.

[0008] DE 10 2017 203 492 A1 (WITec) describes a method and device for imaging a sample surface with a topography using confocal microscopy. The sample is measured using a first laser light source in a Raman or fluorescence measurement. The angular distribution of an incident auxiliary laser beam from a second laser light source is periodically varied using an electrically adjustable lens, and the thus modulated laser beam is directed onto the sample via a beam splitter and the microscope objective. Light from the second light source, reflected from the sample, is directed through the microscope objective and the beam splitter to a point detector, and its power is analyzed. This detector signal is used for readjustment to keep the sample in the focal plane for the fluorescence or Raman measurement.The first light source and the second light source have non-overlapping wavelength ranges. A disadvantage of this approach is the comparatively complex setup required by the second light source. Furthermore, avoiding an overlap between the wavelength range of the second laser light source and the wavelength range of the first laser light source or the excitations generated in the sample is often difficult. Furthermore, the working range is limited due to the necessary distance between the electrically adjustable lens and the microscope objective.

[0009] US 10 067 058 B1 (Renishaw), which is considered to represent the closest prior art, discloses an autofocus system with which, in particular, Raman spectroscopy measurements can be performed on a sample to create a spectroscopic image of the sample. Excitation light from an excitation laser source is focused onto the sample via two beam splitters and a microscope objective. Light returning from the sample is directed via the microscope objective and the beam splitter closer to the sample, firstly via an autofocus lens, a mask with two eccentric apertures and a beam deflection wedge in front of one of the apertures, and a refocusing lens to a spatially resolving detector, and secondly via the further beam splitter to a spectrometer.From the positions of the partial beams generated from the first part on the spatially resolved detector, a control signal is generated to track a sample holder in front of the microscope objective. The disadvantage of this approach is that determining the positions of the partial beams with the spatially resolved detector is error-prone and slow. Light reflected from the sample must pass through the mask apertures with sufficient intensity, which may not be possible, especially if the sample is tilted significantly. In addition, spatially resolved detectors often have only narrow acceptance ranges for laser power, usually by a factor of 8; in Raman spectroscopy, depending on the sample requirements, excitation laser powers that differ by up to a factor of 100 are required, so optical filters must be installed.In addition, the working area is limited due to the necessary distance between the lenses of the autofocus system and the microscope objective.

[0010] DE 10 2017 217 320 B4 (Mitutoyo) describes a lens system with variable focal length and focus control. A light source generates an output light, which is directed onto a workpiece via a partial mirror and through an objective lens. The resulting workpiece light passes through the objective lens, the partial mirror, a tube lens, and a relay lens, then passes through a beam splitter, a lens with variable focal length, another beam splitter, and another relay lens, which directs the workpiece light onto a detector. A control light source is also provided, which couples a focus detection light into the system via the beam splitter. The focus detection light then also passes through the lens with variable focal length and is decoupled at the beam splitter and directed onto a focus control section, which includes a focus photodetector.

[0011] DE 10 2019 219 506 A1 (Mitutoyo) describes a method for calibrating a variable focal length lens system using a calibration object with a flat, inclined sample surface. Workpiece light from a workpiece passes through several lenses, including a variable focal length lens, and is registered by a detector.

[0012] Description of the invention

[0013] The object of the invention is to present a confocal microscope in which automatic tracking of the distance between the sample and the microscope objective is possible in a simple and reliable manner.

[0014] Description of the invention

[0015] This object is achieved according to the invention by a confocal microscope for

[0016] Imaging of a sample, in particular the surface of the sample, comprising an excitation light source, in particular an excitation laser, for generating an excitation light beam, an excitation light beam splitter for coupling the excitation light beam, a point detector for registering sample light caused by the excitation light beam and returning from the sample, in particular from the surface of the sample, a microscope objective for focusing the excitation light beam onto the sample, in particular onto the surface of the sample, and for collecting the sample light returning from the sample, detector optics for imaging the sample light returning from the sample and collected by the microscope objective onto the point detector, a movement device for adjusting a relative distance between the microscope objective and the sample according to a distance control signal, an analysis device,comprising an analysis beam splitter for coupling out a portion of the sample light returning from the sample, an analysis optic for focusing the outcoupled portion of the sample light toward an analysis detector, and the analysis detector for detecting the outcoupled portion of the sample light, wherein the analysis device is configured to generate the distance control signal for the movement device, further wherein the analysis device comprises a modulation device with which the refractive power of the analysis optic and / or a relative distance between the analysis detector and the analysis optic can be cyclically varied in time according to a modulation signal, wherein the analysis device is configured to determine, in a respective modulation cycle of the modulation signal, a focus time at which the outcoupled portion of the sample light is focused on the analysis detector based on a detector signal of the analysis detector,and wherein the analysis device is designed to generate the distance control signal for the movement device by means of the focus time in the respective modulation cycle.,

[0017] overview

[0018] The present invention proposes a confocal microscope in which the excitation light is used both to measure the sample and to track the sample (particularly the local sample surface) relative to the microscope objective. The analysis device (also called the focus finder assembly) is used to control the relative tracking, acting on an outcoupled portion of the sample light returning from the sample.

[0019] A portion of the sample light returning from the sample is coupled out using an analysis beam splitter, directed to an analysis optics, modulated by a modulation device, and detected by an analysis detector to generate a distance control signal. The remaining portion of the sample light returning from the sample is directed to a point detector to obtain information about the sample. This remaining portion of the sample light does not enter the analysis optics and is not modulated by the modulation device.

[0020] The modulation device changes the focus location of the extracted portion of the sample light relative to the analysis detector. Accordingly, a cyclically varying counter-compensation is performed for any momentary defocusing of the excitation light focus relative to the sample (or sample surface). The analysis detector can then determine the currently correct counter-compensation, and from this, the distance control signal for tracking the sample relative to the microscope objective can be obtained.

[0021] Besides the excitation light source, no additional light source is required, which keeps the setup simple and prevents interference with the actual sample measurement due to overlapping wavelength ranges from the outset. The outcoupled portion of the sample light can essentially be used in its entirety during analysis, increasing the reliability of the method. The outcoupled portion originates from sample light generated with the complete excitation light beam, without partial shadows or preferred angles at the sample. Furthermore, the outcoupled portion is essentially generated from the sample light returning from the sample, without partial shadows or preferred directions, and is further processed during analysis. This significantly improves the method's tolerance to any sample tilt, especially severe sample tilt in certain directions.

[0022] The analysis detector does not require a spatial resolution function and is therefore preferably selected as a zero-dimensional detector. The analysis detector can easily be selected with a wide acceptance range (Pmax / Pmin), for example, Pmax / Pmin>50 or Pmax / Pmin>100, which is particularly advantageous for Raman measurements.

[0023] The analysis of a decoupled part of the sample light emanating from the sample also facilitates a compact design by using relay optics.

[0024] Measurement procedure

[0025] During a measurement, the sample is moved (scanned) across a certain area perpendicular to the propagation direction of the excitation light beam. Using the distance control signal, the sample can be tracked in the axial direction (propagation direction of the excitation light beam) relative to the microscope objective, thus keeping the local sample surface at a desired distance from the microscope objective, even if the sample surface exhibits significant roughness.

[0026] The analysis device can be used to determine the current angular distribution / divergence of the sample light, or a portion of it, behind the microscope objective. For example, the angular distribution / divergence of the excitation light reflected from the sample or sample surface depends on its current distance from the microscope objective. If the sample surface is in focus of the microscope objective, the sample light being examined is collimated behind the microscope objective; this typically also corresponds to the desired distance setting for imaging / measuring a particular location on the sample.

[0027] Using the analysis device, a portion of the sample light is extracted behind the microscope objective ("extracted portion") and focused with the analysis optics toward the analysis detector in order to examine the angular distribution / divergence of this extracted portion of the sample light. The axial position of the focus behind the analysis optics varies depending on the angular distribution / divergence of the sample light; this variation in location therefore contains the necessary information about the angular distribution / divergence and thus about the defocusing of the confocal microscope. Furthermore, the location of the focus of the examined portion of the sample light behind the analysis optics relative to the analysis detector is varied in a known manner over a certain range by the inventive modulation (change in the refractive power of the analysis optics and / or change in the distance between the analysis detector and the analysis optics) with the modulation device.By reading the analysis detector during a modulation cycle, the (instantaneous) angular distribution / divergence of the sample light behind the analysis optics is effectively determined.

[0028] The confocal microscope or analysis device is calibrated such that, when the angular distribution / divergence of the sample light (and thus the relative distance between the microscope objective and the sample) is set as desired for the respective measurement (typically with the sample surface in the focus of the microscope objective), the distance control signal derived from the focus time in the modulation cycle maintains the current relative distance between the sample and the microscope objective. Otherwise, the distance control signal causes the relative distance to change to the desired distance. Calibration can be used, in particular, to specify a target focus time in the modulation cycle at which the focus time in the modulation cycle should lie, i.e., at which the focus of the decoupled portion of the sample light should fall on the analysis detector (or its input aperture).For this purpose, for example, a calibration sample (typically with a very flat surface) is first brought to the desired relative distance from the microscope objective using a corresponding distance control signal. For example, the distance control signal can be adjusted so that the sample light signal is maximized at the point detector of the confocal microscope. The focus time in the modulation cycle is then determined in this state; this focus time is then saved as the target focus time and can be used for future samples to be examined. Please note that instead of a calibration sample, a sample to be examined can also be used directly to determine the target focus time; this calibration can then also be used for other samples to be examined, if desired. In the case of rapidly changing measurement conditions (e.g.temperature fluctuations), such a calibration can also be repeated for each sample to be examined before the start of the measurement.

[0029] For the confocal microscope, it is also preferably determined in advance how the focus time in the modulation cycle changes quantitatively when the distance control signal changes ("control function"); this control function can, in many cases (particularly when using relay optics) be described with sufficient accuracy in a linear function within the scope of the invention. However, at least the direction in which the distance control signal must be changed in order to shift the focus time in a specific direction in the modulation cycle ("directional knowledge") should be determined qualitatively. The control function or directional knowledge are essentially system properties of the confocal microscope, which generally do not need to be re-determined during calibrations.

[0030] During subsequent measurement / imaging of samples, the distance control signal can now be adjusted or tracked at each measurement location so that the current focus time corresponds to the stored target focus time. This ensures that the sample (or its local sample surface) is also at the desired relative distance from the microscope objective at the current measurement location. When adjusting / tracking the distance control signal, knowledge of the control function, or at least knowledge of the direction, can be used.

[0031] It should be noted that the target focus time corresponds to a target focus elongation of the modulation signal, and accordingly, these variables carry equivalent information about the target distance between the sample and the microscope objective. Likewise, the (current) focus time corresponds to a (current) focus elongation of the modulation signal, and accordingly, these variables carry equivalent information about the current distance between the sample and the microscope objective. In practice, against this background and in accordance with the invention, one variant provides that the (current) focus elongation of the modulation signal is determined via the (current) focus time in the modulation cycle. For example, in a modulation cycle, the modulation signal (e.g., the control signal / control voltage of an ETL) changes continuously over time between a minimum and a maximum (e.g., sinusoidally).At the focus time, the modulation signal is read out, thus obtaining the focus elongation. The distance control signal can then be determined from the focus elongation, particularly by comparing it with the target focus elongation.

[0032] Note that a height profile of the sample surface can be created using the distance control signal as a function of the grid location.

[0033] Beam splitter arrangements

[0034] Various arrangements are available for the excitation light beam splitter for coupling the excitation light beam and the analysis beam splitter for coupling out the sample light. Generally, the excitation light beam splitter is positioned between the microscope objective and the detector optics.

[0035] In a short-throw (KD) arrangement of the two beam splitters, both beam splitters are positioned between the sample and the point detector, typically between the microscope objective and the detector optics. For example, the analysis beam splitter can be positioned between the microscope objective and the excitation light beam splitter, and the excitation light beam splitter can be positioned between the analysis beam splitter and the detector optics. Separate, parallel beam paths are set up for the excitation light beam to be coupled in and the portion of the sample light to be coupled out. The KD arrangement is characterized by a particularly short optical path length between the microscope objective and the analysis device.

[0036] Both beam splitters can also be arranged in a long-distance (LD) configuration, allowing more sample light to reach the point detector, as the sample light only needs to pass through the excitation light beam splitter on its way to the point detector. In the LD configuration, only the excitation light beam splitter is positioned between the microscope objective and the detector optics, not the analysis beam splitter. The analysis beam splitter is located between the excitation light source and the excitation light beam splitter. The incoming excitation light beam and the outgoing portion of the sample light travel an identical partial beam path between the excitation light beam splitter and the analysis beam splitter.

[0037] In a special case of the KD arrangement, the analysis beam splitter and the excitation light beam splitter can also be combined in a wedged beam splitter. A wedge creates a reflection on the front and back, thus creating two separate optical paths. One path then serves to reflect the excitation light toward the microscope objective, while the other path couples the sample light into the analysis optics.

[0038] Aspects of excitation light and sample light and other aspects The excitation light preferably comes from a monochromatic excitation laser.

[0039] The sample light comprises elastically backscattered excitation light (reflection light) from the sample and / or fluorescence light generated by the excitation light in the sample and / or inelastically backscattered excitation light (Raman light). The elastically backscattered excitation light has the same frequency (or the same frequency spectrum) as the excitation light. Fluorescence light and Raman light are frequency-shifted relative to the excitation light. Depending on the portion of the sample light evaluated by the point detector, the confocal microscope can be used for confocal light microscopy, confocal fluorescence microscopy, or confocal Raman microscopy.

[0040] The sample light, generated by the excitation laser beam and returning from the sample, is used to image the sample and is characteristic of the local sample properties. Fluorescence light and / or Raman light can be used to determine the local chemical composition of the sample; reflected light can at least be used to determine the local reflectivity (i.e., the "brightness" or, in the case of a spectrally resolved measurement, the "color") of the sample. The point detector can be designed, in particular, as a spectrograph with a (confocal) pinhole as the input aperture.

[0041] The analysis beam splitter and the excitation light beam splitter typically have different reflection properties. The excitation light beam splitter typically consists of an optical filter, such as a bandpass filter, which spectrally separates the sample light for use in the point detector from the elastically backscattered excitation light (particularly for Raman or fluorescence microscopy). The optical filter typically blocks the transmission of the excitation light. The analysis beam splitter is typically an intensity beam splitter, such as a glass plate, which reflects a small portion of the sample light (approximately spectrally independent).The analysis device typically utilizes the elastically backscattered excitation light (reflection light) in the sample light; for the method according to the invention, even a comparatively small amount of light reflected back into the confocal microscope is sufficient, which particularly improves tolerance to sample tilt. The proportion of reflected light generally dominates the sample light. The analysis beam splitter has a reflectivity of typically 10% or less, preferably 5% or less, for coupling the portion of the sample light returning from the sample to the analysis optics and the analysis detector. Then, the excitation light beam is only slightly attenuated on the way to the sample and / or the sample light on the way to the point detector, and a correspondingly high intensity of sample light is available for measuring / imaging the sample.

[0042] To adjust the relative distance between the microscope objective and the sample, the movement device can move the sample in the direction of propagation of the excitation light beam, particularly with a stationary microscope objective, or alternatively, with a stationary sample, move the microscope objective in the direction of propagation of the excitation light beam (movement in z). Preferably, the movement device can also be used to move the sample perpendicular to the beam propagation direction relative to the microscope objective for scanning (movement in x, y); alternatively, a further, independent movement device can also be provided for scanning.

[0043] Preferred embodiments of the invention

[0044] In a preferred embodiment of the confocal microscope, the analysis device is configured to indirectly generate the distance control signal for the movement device using the focus time in the respective modulation cycle. This is done by reading out an elongation of the modulation signal at the focus time, and generating the distance control signal for the movement device using this elongation of the modulation signal at the focus time. The elongation of the modulation signal at the focus time, also called focus elongation, contains particularly precise information about the current distance between the sample and the microscope objective, thus enabling particularly precise tracking of the sample.

[0045] A preferred embodiment of the confocal microscope according to the invention is one in which the analysis optics comprise an electrically adjustable lens whose refractive power can be cyclically varied over time by means of the modulation signal. The electrically adjustable lens allows the focus location of the outcoupled portion of the sample light to be changed relative to the analysis detector. This is structurally simple and cost-effective, and allows particularly rapid determination of the current focus time (and thus indirectly the focus elongation) within the cycle of the modulation signal. Good mechanical stability of the confocal microscope as a whole can also be achieved. In this embodiment, both the analysis optics and the analysis detector are typically stationary. The electrically adjustable lens is typically formed by a fluid-filled volume that is bounded on at least one side by a membrane.This membrane can be deformed by a voice coil so that a desired lens curvature and thus refractive power is achieved.

[0046] In a preferred development of this embodiment, a modulation frequency of the modulation signal corresponds to a natural frequency of the electrically adjustable lens. In this case, particularly large adjustment ranges of the refractive power can be achieved.

[0047] A further development is particularly preferred which provides that the analysis optics comprise the electrically adjustable lens on the input side and a non-adjustable, imaging optical element, in particular a non-adjustable lens, on the output side. The adjustment range of the analysis optics or the distance tracking range of the confocal microscope can be shifted using the non-adjustable, imaging optical element (usually a fixed lens). Typically, the adjustment range of the analysis optics is shifted so that it lies symmetrically around the desired focal point. A further development in which the electrically adjustable lens is aligned horizontally is preferred. In this case, distortions on the membrane of the electrically adjustable lens caused by gravity are minimized, in particular asymmetrical distortions are minimized.

[0048] In an alternative embodiment, the analysis detector is arranged on a wobble device, with which the position of the analysis detector can be cyclically changed in time in the propagation direction of the decoupled part of the sample light, in particular with the analysis optics being stationary. The wobble device can also be used to change the location of the focus of the decoupled part of the sample light relative to the analysis detector. With the wobble device, the determination of the current focus time (and thus the current focus elongation) is typically somewhat slower than with an electrically adjustable lens; however, the wobble device is usually less sensitive to any high-frequency vibrations than an electrically adjustable lens. In this embodiment, the refractive power of the analysis optics is typically not adjustable.

[0049] Embodiments relating to a relay optic

[0050] Particularly preferred is an embodiment which provides that a relay optic is arranged between the analysis beam splitter and the analysis optics, with which the exit pupil of the microscope objective is imaged onto an entrance pupil of the analysis optics with an imaging factor ABF. The relay optics can be used to shift the compensable detuning range and / or to enlarge the maximum compensable detuning range of the distance between the sample and the microscope optics. In particular, the relay optics can be used to prevent the focus of the decoupled part of the light from passing through the location of an electrically adjustable lens of the analysis optics beyond the desired detuning range of the confocal microscope, which would make the directional knowledge (see above) for tracking the distance between the sample and the microscope objective in the desired detuning range no longer clear.It can also be ensured that the focus of the decoupled part also reaches the position of the analysis detector (or its entrance aperture) across the desired detuning range, for example, in the center of the desired detuning range. When the exit pupil of the microscope objective is imaged or effectively transferred to the entrance pupil of the analysis optics using the relay optics, the angular distribution of the light returning from the sample is changed according to the image ratio.

[0051] A preferred development of this embodiment provides that the relay optics comprises an even number N of imaging element groups, wherein each imaging element group comprises at least one imaging optical element, wherein each imaging element group i can be assigned a front focal length FFL_i, a back focal length BFL_i and an effective focal length EFL_i, where i: index of the imaging element groups, and that at least approximately the exit pupil of the microscope objective is positioned at a distance FFL_1 in front of the first imaging element group i = l, and at least approximately the entrance pupil of the analysis optics is positioned at a distance BFL_N behind the last imaging element group i = N, and adjacent element groups i, i + 1 are at least approximately positioned at a distance d(i,i + 1) = BFL_i + FFL_i + 1 from one another. In this way, the relay optics can be set up in a simple manner.In many applications, exactly two imaging element groups are set up, i.e. N=2. The imaging factor ABF can be determined via the ratio of the effective focal lengths. In the case of two imaging groups (with indices 1 and 2), the imaging factor can be determined via the effective focal lengths: ABF=EFL_2 / EFL_1. The imaging optical elements can be refractive and / or diffractive and / or reflective and / or comprise meta-optics. The imaging optical elements can comprise lenses, in particular convex lenses and / or concave lenses, and / or curved mirrors, in particular concave mirrors. Please note that small variations in the aforementioned positions of the exit pupil or entrance pupil or said distances (on the order of e.g. up to 2 mm, which corresponds roughly to the axial expansion range when adjusting an electrically adjustable lens) are not critical for the relay optics.

[0052] A preferred further development of this embodiment is one in which at least one imaging element group comprises at least two imaging optical elements. This allows for more complex beam paths and, if necessary, also stronger light refraction in the imaging element group.

[0053] A particularly preferred development is one in which the first imaging element group comprises at least two imaging optical elements, and these imaging optical elements are selected such that FFL_1>BFL_1. This allows a particularly compact design of the relay optics in the beam propagation direction to be achieved. For this purpose, the first imaging element group can, for example, contain a convex lens followed by a concave lens / diffuser lens, which together, firstly, establish a first front focal length FFL_1 that is sufficiently long for the measurement setup and, secondly, shorten the first back focal length BFL_1.

[0054] In an advantageous development of the above embodiment, the imaging factor ABF is 1. This is particularly easy to set up; in particular, this "direct" transfer can be set up with a total of only two lenses (so-called 4f correlator), i.e., with two identical imaging element groups, each with only one imaging optical element.

[0055] In an alternative, likewise preferred embodiment, the imaging factor ABF is magnifying, with ABF > 1, in particular where 1 < ABF < 3. The magnifying image can, in many cases, reduce the necessary adjustment range of the refractive power of a downstream analysis optic (or the travel range of the analysis detector) in order to cover a predetermined detuning range of the sample / sample surface. The downstream analysis optic must have an entrance pupil sufficiently large for the imaging factor. Further embodiments, uses, and methods

[0056] A further preferred embodiment of the confocal microscope provides that the analysis detector has an entrance aperture with a diameter no larger than the diameter of the portion of the sample light fully focused by the analysis optics, and that the analysis device is configured to determine the focus time by ensuring that the intensity of the detector signal in the modulation cycle is maximum at the focus time. Such a sufficiently small entrance aperture ensures a clearly detectable, sharp maximum of the intensity of the detector signal when the focus of the decoupled portion of the sample light falls precisely on the location of the entrance aperture of the analysis detector, and the focus time can be determined with high accuracy, <10 microseconds, preferably <1 microsecond. The sample can then be measured with corresponding precision.The diameter of the focus (perpendicular to the beam propagation direction) is then determined using the 86% criterion, i.e. 86% of the beam power of the outcoupled part of the sample light lies within a circle with the said diameter.

[0057] The scope of the present invention also includes the use of a confocal microscope according to the invention, as described above, for imaging a sample, in particular a surface of the sample, by means of confocal microscopy, in particular confocal light microscopy, confocal Raman microscopy or confocal fluorescence microscopy, wherein the sample is measured successively at a plurality of measuring locations, wherein in order to change the measuring location the sample is moved transversely to the propagation direction of the excitation light beam, and wherein the relative distance between the microscope objective and a local surface of the sample at the respective measuring location is kept at least approximately constant across the various measuring locations using the movement device using the distance control signal generated by the analysis device.This allows a uniformly good focus of the confocal microscope on the sample to be achieved in a simple and reliable manner in the intended imaging area of ​​the sample, which is scanned by the measuring locations.

[0058] Also within the scope of the present invention is a method for imaging a sample, in particular the surface of the sample, using confocal microscopy, in particular using confocal light microscopy, confocal Raman microscopy or confocal fluorescence microscopy, and in particular wherein the method is carried out on a confocal microscope according to the invention, as described above, comprising the following steps:

[0059] An excitation light source, in particular an excitation laser, generates an excitation light beam,

[0060] The excitation light beam is coupled in using an excitation light beam splitter;

[0061] A point detector registers the sample light coming back from the sample, especially from the surface of the sample, caused by the excitation laser beam.

[0062] With a microscope objective, the excitation light beam is focused onto the sample, especially onto the surface of the sample, and the sample light returning from the sample is collected,

[0063] Using detector optics, the collected sample light returning from the sample is imaged onto the point detector;

[0064] A movement device is used to adjust a relative distance between the microscope objective and the sample according to a distance control signal,

[0065] The distance control signal for the movement device is generated by an analysis device comprising an analysis beam splitter that couples out a portion of the sample light returning from the sample, an analysis optic that focuses the decoupled portion of the sample light toward an analysis detector, and the analysis detector that detects the decoupled portion of the sample light. The analysis device further comprises a modulation device with which the refractive power of the analysis optic and / or a relative distance between the analysis detector and the analysis optic is cyclically changed in time according to a modulation signal. The analysis device determines a focus time at which the decoupled portion of the sample light is focused on the analysis detector in a respective modulation cycle of the modulation signal based on a detector signal of the analysis detector.and wherein the analysis device generates the distance control signal for the movement device using the focus time in the respective modulation cycle. Using the method according to the invention, a sample can be measured simply and reliably using confocal microscopy.

[0066] In a preferred variant of the method according to the invention, the point detector is designed as a spectrograph, and a spectral composition of the light returning from the sample is analyzed using the spectrograph. Fluorescence properties and / or Raman scattering properties of the sample are determined in the sample light returning from the sample. In particular, sample light reflected from the sample, which has the same wavelength as the excitation light beam, is blocked upstream of the point detector using a filter (optionally with multiple filter stages). Within the scope of the invention, fluorescence and Raman microscopy with a good signal-to-noise ratio are possible, particularly when different intensities of the excitation light must be used to determine different spectral lines.For example, in the case of a Raman microscope, the excitation beam splitter can be selected so that it already provides an initial spectral separation of the excitation light from the Raman-scattered light.

[0067] Further advantages of the invention will become apparent from the description and the drawings. Likewise, the above-mentioned and further-described features can be used individually or in combination in any desired manner. The embodiments shown and described are not intended to be exhaustive, but rather are exemplary in nature for describing the invention.

[0068] Detailed description of the invention and drawing

[0069] Fig. 1 shows a schematic representation of a first embodiment of a confocal microscope according to the invention, with beam splitters in KD configuration;

[0070] Fig. 2 shows a schematic representation of a second embodiment of a confocal microscope according to the invention, with beam splitters in LD configuration;

[0071] Fig. 3 shows a schematic representation of a third embodiment of a confocal microscope according to the invention, with a wedge-shaped beam splitter (and KD configuration);

[0072] Fig. 4 shows schematically a beam path of the sample light in confocal microscopy in front of a detector (point detector or analysis detector), at different distances from the sample and the microscope objective, for the invention;

[0073] Fig. 5 shows schematically a beam path of the sample light in front of the analysis detector, with different refractive power of an electrically adjustable lens, for the invention;

[0074] Fig. 6 schematically shows a beam path of the sample light in front of the analysis detector, which is arranged on a wobbler, for the invention; Fig. 7 schematically shows a beam path of the sample light in an analysis device without relay optics, for a first distance between the sample and the microscope objective, for the invention;

[0075] Fig. 8 shows schematically a beam path of the sample light in an analysis device without relay optics, for a second distance between sample and microscope objective, for the invention;

[0076] Fig. 9 shows schematically the beam path of the sample light similar to Fig. 7, in an analysis device with relay optics, for the first distance between sample and microscope objective, for the invention;

[0077] Fig. 10 shows schematically the beam path of the sample light similar to Fig. 8, in an analysis device with relay optics, for the second distance between sample and microscope objective, for the invention;

[0078] Fig. 11 shows schematically a beam path of the sample light in the region of a relay optics for the invention, which is constructed as a 4f correlator;

[0079] Fig. 12 shows schematically a beam path of the sample light in the region of a relay optics for the invention, which provides imaging with ABF=1, and wherein a first element group comprises two imaging optical elements;

[0080] Fig. 13 shows schematically a beam path of the sample light in the region of a relay optics for the invention, which provides imaging with ABF=2, and wherein a first element group comprises two imaging optical elements;

[0081] Fig. 14 shows a schematic diagram of a modulation signal over half a modulation cycle, with the value M of the modulation signal plotted as a function of time t, for the invention; Fig. 15 shows a schematic diagram of a detector signal, with signal strength D of the detector signal at an analysis detector as a function of time, with sample detuning, for the invention.

[0082] Fig. 1 shows a schematic representation of a first embodiment of a confocal microscope 1 according to the invention for imaging a sample 2. The sample 2 is arranged on a sample holder 3, which can be moved in three orthogonal spatial directions x, y, z; the movable sample holder 3 is also referred to as the movement device 3 of the sample 2. The sample 2 has a roughness in the direction toward the confocal microscope 1 or its microscope objective 4, i.e., in the z direction. To image the sample 2, different locations on the sample 2 are measured sequentially ("measurement locations"), for which the sample holder 3 is moved (scanned) in x and y axes. A (local) distance AB between the sample 2 or its local surface and the microscope objective 4 is kept constant across all measurement locations, for which the sample holder 3 is adjusted in z according to a distance control signal.The sample holder 3 is controlled by an electronic control device 23.

[0083] An excitation light source 5, here a monochromatic excitation laser with an output aperture 5a, generates an excitation light beam 6, which is collimated here by a lens 5b and coupled into the confocal microscope 1 via an excitation light beam splitter 7. The excitation light beam 6 is reflected downward by the excitation light beam splitter 7 at its lower boundary surface in the embodiment shown, passes through an analysis beam splitter 8, and is focused by the microscope objective 4 onto the (local) surface of the sample 2, cf. the sample-side beam focus 9.

[0084] The excitation light beam 6 is partially elastically scattered (generating reflected light), partially inelastically scattered (generating Raman light) at the sample 2 in the area of ​​the sample-side beam focus 9, with proportions depending on the sample material, and partially fluorescence is excited in the sample 2 (generating fluorescent light). In summary, sample light 10 is generated at the sample 2 and returned to the confocal microscope 1.

[0085] The sample light 10 returning from the sample 2 is collimated at the microscope objective 4 (assuming correct focus). A portion of the sample light 10 is coupled out at the analysis beam splitter 8 (see the coupled-out portion 11a) and directed to an analysis optics 12. The position of the analysis beam splitter 8 shown in Fig. 1 enables a particularly short optical path from the microscope objective 4 to the analysis optics 12 and is therefore called a short-throw configuration (KD configuration). A non-coupled portion 11b of the sample light 10 reaches the excitation light beam splitter 7. This is designed here as an optical filter that blocks the wavelength (or wavelength range) of the excitation light beam 6. For illustrative purposes, the representation of the light beams is simplified, particularly at both beam splitters, and shown without the effects of optical refraction (this also applies to the other figures).A remaining portion 11c of the sample light 10, which is dominated by fluorescence and / or Raman light from the sample 2, passes through the excitation light beam splitter 7 and reaches a detector optics 13, where it is focused by the detector optics 13 onto an entrance aperture 14a of a point detector 14. The point detector 14 is wavelength-sensitive, i.e., a spectrograph 14b.

[0086] A high intensity of the sample light 10 originating from the sample surface of the sample 2 is only obtained at the point detector 14, or the point detector-side beam focus 15 of the remaining part 11c of the sample light 10 is only located in the axial direction (z-direction) at the location of the entrance aperture 14a of the point detector 14, if the sample-side beam focus 9 is also located exactly on the surface of the sample 2 ("confocal" geometry). In this case (as shown in Fig. 1), the sample light 10 is also collimated between the microscope objective 4 and the detector optics 13. If, for example, the sample-side beam focus 9 is located above the local sample surface in the z-direction due to the roughness of the sample 2 (i.e., the sample has a local "valley", oris currently "too far away" from the microscope objective 4), a converging beam of sample light (11b) is obtained, and if the sample-side beam focus 9 in z lies below the local sample surface (i.e. the sample 2 has a local "mountain" or is currently "too close" to the microscope objective 4), a divergent beam of sample light (11b) is obtained, in each case seen from the microscope objective 4 in the direction of the analysis beam splitter 8.

[0087] In order to always keep the current distance AB of the sample 2 or the local sample surface from the microscope objective 4 at the desired distance (also called target distance ABsoll, not shown separately), according to the invention an analysis device 16 uses the decoupled part 11a of the sample light to carry out an analysis which results in the generation of the distance control signal with which the distance AB of the sample 2 from the microscope objective 4 can always be kept at the desired local distance ABsoll.

[0088] The analysis device 16 comprises the analysis beam splitter 8, the analysis optics 12, an analysis detector 17, a modulation device 22 and the electronic control device 23, as well as a relay optics 18 between the analysis beam splitter 8 and the analysis optics 12. The analysis optics 12 here comprises an electrically adjustable lens 19 (also called ETL, electrically tunable lens) and a non-adjustable lens 20. The analysis detector 17 is designed, for example, as a photodetector, in particular a zero-dimensional photodetector.

[0089] The analysis beam splitter 8 reflects approximately 5% of the total intensity of the sample light 10 as the outcoupled portion 11a toward the analysis optics 12, approximately independent of the wavelength; the outcoupled portion 11a is accordingly dominated by reflected light. The outcoupled portion 11a of the sample light 10 is focused by the analysis optics 12 toward the analysis detector 17, cf. the analysis detector-side beam focus 21. The position of the beam focus 21 depends on the current distance AB of the sample 2 or the local sample surface from the microscope objective 4.

[0090] With the modulation device 22, the position of this beam focus 21 in the axial direction (beam propagation direction of the decoupled part 11a, here x-direction) relative to the analysis detector 17 or its input aperture 17a is cyclically changed ("modulated") over time, here by changing the refractive power of the electrically adjustable lens 19. The modulation device 22 generates a modulation signal for this purpose, which here controls the lens curvature of the ETL 19 as an electrical voltage on a voice coil (not shown in detail).

[0091] Over a modulation cycle, the analysis detector-side beam focus 21 briefly reaches the axial position of the input aperture 17a of the analysis detector 17, thereby maximizing its detector signal. From the time at which this maximum value of the detector signal is reached in the modulation cycle or from the associated elongation (also called focus elongation) of the modulation signal, compared to a calibrated, desired target time or a calibrated target elongation (also called target focus elongation) in the modulation cycle, the electronic control device 23 can determine the current detuning of the sample distance AB relative to the desired distance ABsoll, and generate a suitable distance control signal for tracking the sample 2 to the desired distance ABsoll (see also Fig. 14 and Fig. 15).

[0092] Fig. 2 shows a second embodiment of a confocal microscope 1 according to the invention, similar to the confocal microscope of the first embodiment of Fig. 1. Therefore, only the essential differences are explained, and for the sake of simplicity, some of the same elements are not shown again.

[0093] In the confocal microscope 1 of Fig. 2, only the excitation light beam splitter 7 is arranged in the beam path between the microscope objective 4 and the detector optics 13, but not the analysis beam splitter 8. Rather, the analysis beam splitter 8 is arranged between the excitation light source 5 and the excitation light beam splitter 7. The excitation light beam 6 passes through the analysis beam splitter 8 and is reflected by the excitation light beam splitter 7 at its lower interface in the direction of the microscope objective 4. Sample light 10 emanating from the sample 2 is reflected with a first part 11d toward the analysis beam splitter 8, and a remaining part 11c passes through the excitation light beam splitter 7, which in turn acts as an optical filter and blocks the wavelength of the excitation light. From the first part 11d, an outcoupled part 11a is reflected by the analysis beam splitter 8 in the direction of the analysis optics 12.

[0094] With this design, a particularly high proportion of the sample light 10 can reach the point detector 14. Due to the comparatively long optical path from the microscope objective 4 to the analysis optics 12, this design is called a long-distance configuration (LD configuration).

[0095] Fig. 3 shows a third embodiment of a confocal microscope 1 according to the invention, similar to the confocal microscope of the first embodiment of Fig. 1. Therefore, only the essential differences are explained, and for the sake of simplicity, some of the same elements are not shown again.

[0096] In the embodiment of Fig. 3, the excitation light beam splitter 7 and the analysis beam splitter 8 are combined in a common component, namely a beam deflection wedge 30 (also called a wedged beam splitter). An upper boundary surface of the beam deflection wedge 30 acts as the excitation light beam splitter 7, which in particular couples the excitation light beam 6 from the excitation light source 5 and reflects it toward the microscope objective 4. This upper boundary surface of the beam deflection wedge 30 can be designed as an optical filter that blocks the wavelength (or wavelength range) of the excitation light beam 6 by virtually completely reflecting this wavelength. A lower boundary surface of the beam deflection wedge 30 acts as an analysis beam splitter 8 and, in particular, couples out an outcoupled part 11a of the sample light 10 emanating from the sample 2 and reflects this outcoupled part 11a in the direction of the analysis optics 12.The material of the beam deflection wedge 30 is typically transparent to the wavelengths of the excitation light beam 6 and the sample light 10.

[0097] This design allows for a particularly compact construction, and this design also corresponds to a KD configuration. Figures 4 to 6 schematically illustrate various influences on the position of a beam focus in a confocal microscope according to the invention.

[0098] Fig. 4 shows schematically and by way of example the beam path of the coupled-out part 11a of the sample light near a last lens 41 of an analysis optics 12 in front of an analysis detector 17. It should be noted that analogous conditions apply at the last lens of a detector optics in front of a point detector for the measurement of the remaining part of the sample light.

[0099] When the distance between the sample and the microscope objective is focused, the outcoupled portion 11a is collimated in front of the lens 41, and its focus 42 is precisely imaged onto the axial location of an entrance aperture 17a of the analysis detector 17 (solid lines). In this situation, the detector signal of the analysis detector 17 is at its maximum.

[0100] However, if the sample is "too far away" from the microscope objective, the outcoupled part 11a is convergent in front of the lens 41, and its focus 43 lies axially in front of the entrance aperture 17a (dashed lines). In this situation, a detector signal of the analysis detector 17 is reduced.

[0101] If, however, the sample is "too close" to the microscope objective, the outcoupled beam 11a is divergent in front of the lens 41, and its focus 44 lies axially behind the entrance aperture 17a (dotted lines). In this situation, a detector signal of the analysis detector 17 is also reduced.

[0102] According to the invention, the focus position of the outcoupled part 11a is adjusted by means of a modulation device 22, which, in Fig. 5, can adjust the refractive power of an electrically adjustable lens 19. The electrically adjustable lens 19 is the last lens of the analysis optics 12 here; however, it should be noted that the same would also apply if the electrically adjustable lens 19 were, for example, the second-to-last lens of the analysis optics 12 (as shown in Fig. 1). For simplicity, it is assumed in Fig. 5 that the outcoupled part 11a is collimated; however, the explained shift of the focus 45 also applies accordingly for a divergent or convergent outcoupled part 11a.

[0103] At medium refractive power of the ETL 19 (solid lines), in the situation shown in Fig. 5, the focus 45 of the decoupled part 11a lies on the axial position of the input aperture 17a of the analysis detector 17. In this situation, a detector signal of the analysis detector 17 is again at a maximum.

[0104] With increased refractive power of the ETL (dashed lines), the focus 45 is moved forward, and accordingly the focus 46 is now in front of the entrance aperture 17a.

[0105] When the refractive power of the ETL is reduced (dotted line), the focus 45 is moved backwards, and accordingly the focus 47 of the decoupled part 11a is then behind the entrance aperture 17a.

[0106] If there is a misalignment of the distance between the sample and the microscope objective compared to the focus (see Fig. 4), and the refractive power of the ETL 19 is then varied cyclically over time using the modulation signal from the modulation device 22, as shown in Fig. 5, the focus 45 / 46 / 47 of the outcoupled portion 11a of the sample light briefly reaches the axial position of the input aperture 17a. This occurs precisely when the adjustment of the refractive power of the ETL 19 from the mean refractive power precisely compensates for the misalignment of the distance between the sample and the microscope objective compared to the focus. This point in time in the modulation cycle, or the associated elongation of the modulation signal in the modulation cycle, can be determined based on the maximum intensity of the detector signal in the modulation cycle, and from this, a distance control signal can be obtained with which the momentary misalignment of the sample distance can be eliminated.

[0107] It should be noted that the focus shift due to changing the refractive power and the detuning of the sample are monotonically related, so that a monotonic control function can be obtained. Preferably, the focus 45 / 46 / 47 of the decoupled part 11a can be adjusted symmetrically around the center position of the focus 45, which corresponds to the focusing position, by means of the modulation device 22.

[0108] Instead of adjusting the refractive power of an ETL, the position of the analysis detector 17 or the position of its entrance aperture 17a relative to the analysis optics 12 can also be adjusted according to the invention. In other words, the relative distance RA between the analysis optics 12 and the analysis detector 17 can be changed. The latter is illustrated schematically and by way of example in Fig. 6. The analysis detector 17 is arranged here on a wobble device 48, with which the analysis detector 17 can be moved cyclically in time between a front position (shown in dashed lines) and a rear position (shown in dotted lines), in accordance with a modulation signal from the modulation device 22.

[0109] Preferably, by means of the modulation device 22, the (relative) position of the analysis detector 17 can be adjusted symmetrically around the central position of the analysis detector 17 (shown in solid line) belonging to the focus.

[0110] Figures 7 to 10 illustrate the benefits of relay optics that can be used within the scope of the invention. The figures each illustrate the beam path of the outcoupled portion 11a of the sample light in the region from the microscope objective 4 to slightly behind an electrically adjustable lens 19 of an analysis device according to the invention (see Fig. 1 for this purpose); beam deflections by beam splitters and the beam splitters themselves are omitted for simplicity.

[0111] In a simple design of the analysis device, shown in Fig. 7, it is designed without relay optics. When focused, the decoupled part 11a of the sample light would be collimated behind the microscope objective 4 (not shown). With increased distance between the sample and the microscope objective 4, the decoupled part 11a becomes convergent, whereby with slight defocusing the focus 50 remains behind the electrically adjustable lens 19. As long as this is the case, the distance tracking between the sample and the microscope objective 4 (i.e., the control of the sample holder in the z-direction) can still be carried out clearly. For samples with low roughness, the invention can accordingly be implemented in a simple manner even without relay optics.

[0112] If the distance between the sample and the microscope objective 4 becomes significantly too large, the focus 51 moves in front of the electrically adjustable lens 19 without further action, as shown in Fig. 8, and distance tracking is no longer possible. It should be noted that, for structural reasons, the ETL 19 cannot be moved as close as desired to the microscope objective 4 (the minimum distance is usually approximately 25 cm due to structural reasons). For example, the analysis beam splitter must be placed between them.

[0113] Within the scope of the invention, a relay optics 90 can then be used, which is placed between the analysis beam splitter and the analysis optics. This images an exit pupil 4a of the microscope objective 4 onto an entrance pupil 12a of the analysis optics. Accordingly, the beam path of the outcoupled portion 11a of the sample light is also imaged, i.e., transferred from the exit pupil 4a to the entrance pupil 12a (if necessary, applying an imaging factor of the relay optics 90).

[0114] The section SCI of the beam path of the decoupled part 11a of Fig. 7 is identically shown in Fig. 9 at the exit pupil 4a. With the relay optics 90, this section SCI is reproduced behind the electrically adjustable lens 19, which here represents the entrance pupil 12a of the analysis optics, and the beam path then continues accordingly (see Fig. 7).

[0115] Likewise, section SC2 of the beam path of the decoupled portion 11a of Fig. 8 is identical to Fig. 10 at the exit pupil 4a. With the relay optics 90, this section SC2 is also reproduced behind the electrically adjustable lens 19, which also represents the entrance pupil 12a of the analysis optics, and the beam path then continues accordingly (see Fig. 8).

[0116] This ensures that the focus 50, 51 (Fig. 7, Fig. 8) of the decoupled portion 11a of the sample light in Fig. 9 and Fig. 10, i.e., when using the relay optics 90, is always located behind the electrically adjustable lens 19, and distance tracking is always possible in a clear manner. The entrance pupil 12a of the analysis optics, or the ETL 19, is then located virtually at the location of the microscope objective 4, which increases the detectable detuning range.

[0117] In the cases shown in Fig. 9 and Fig. 10, the relay optics 90 has only two imaging optical elements E1.1, E2.1, each representing an imaging element group G1, G2 (each with only one imaging optical element). Furthermore, the relay optics 90 has an imaging factor ABF of 1 (more on this below).

[0118] Figures 11, 12 and 13 illustrate various designs of relay optics 90 that can be used for the invention.

[0119] Fig. 11 illustrates a first design of a relay optic 90 that can be used within the scope of the invention, and also explains the most important geometric boundary conditions for a relay optic 90.

[0120] The relay optics 90 here comprises a number of N = 2 imaging element groups G1, G2 (the index of the element groups is also denoted by i, here with i = 1, 2); note that a larger, even number N of element groups can also be provided. Each element group G1 comprises one or more imaging optical elements E1.j, with j: index of the elements in group i. In the design shown, the element group G1 has only the element E1.1, and the element group G2 has only the element E2.1.

[0121] At a distance corresponding to the front focal length FFL_1 of the first group Gl is the entrance plane 101 of the relay optics 90; the exit pupil 4a of the microscope objective is located there. At a distance corresponding to the back focal length BFL_N of the last group GN is the exit plane 102 of the relay optics 90; the entrance pupil 12a of the analysis optics is located here. Note that in the embodiment shown, GN=G2. Successive element groups are spaced at a distance equal to the sum of the back focal length BFL_i of the front element group i and the front focal length FFLJ+1 of the rear element group i + 1. In this case, the element groups Gl, G2 are arranged at a distance d(l,2) = BFL_l + FFL_2. Here, centrally between the element groups Gl, G2 is a Fourier plane 103 with an intermediate focus of the decoupled part 11a.

[0122] In the first design shown in Fig. 11, elements E1.1 and E2.1 have the same focal length, and all four distances (from 101 to E1.1, from E1.1 to 103, from 103 to E2.1, and from E2.1 to 102) are equal. This corresponds to a so-called 4f correlator. The relay optics 90 has an imaging factor ABF of 1, so that the beam width SW1 of the decoupled part 11a at the input plane 101 is equal to the beam width SW2 at the output plane 102.

[0123] Fig. 12 shows another design of a relay optic 90 for the invention, which largely corresponds to the relay optic of Fig. 11, so that only the essential differences are explained.

[0124] In this design, the first element group Gl has two imaging optical elements El.1 and El.2. The first element El.1 is a convex lens, and the second element El.2 is a concave lens. A suitable choice of the lenses and the distance between El.1 and El.2 ensures a large front focal length FFL_1 and a short back focal length BFL_1 of the first element group Gl. Accordingly, FFL_1>BFL_1 applies, with FFL_1>3*BFL_1 being preferred, or FFL_1>10*BFL_1 being particularly preferred. This allows a large distance (along the beam propagation direction) from the microscope objective or its exit pupil 4a to the relay optics 90 to be provided, while at the same time keeping the overall axial length of the relay optics 90 small. The analysis optics and the analysis detector can thus be positioned practically freely, especially in KD and LD configuration, see Fig. 1, Fig. 2 and Fig.3, and in particular without affecting the working range of the detectable detuning of the confocal microscope.

[0125] The relay optics 90 of Fig. 12 also has an imaging factor ABF of 1, so that the beam width SW1 at the input plane 101 is equal to the beam width SW2 at the output plane 102.

[0126] Fig. 13 shows another design of a relay optic 90 for the invention, which largely corresponds to the relay optic of Fig. 12, so that only the essential differences are explained.

[0127] In this design, the element groups Gl, G2 are designed in such a way, and in particular the effective focal lengths EFL_1, EFL_2 of the two groups Gl, G2 are selected in such a way that the relay optics 90 as a whole has an imaging factor ABF=2, so that the beam width SW2 at the output plane 102 is twice as large as the beam width SW1 at the input plane 101, i.e. SW2=2*SW1.

[0128] Using the imaging factor ABF, especially with ABF>1, beam divergence can be balanced against beam size. In particular, magnification (ABF>1) can reduce the required refractive power of a downstream ET L.

[0129] Figures 14 and 15 illustrate schematically and by way of example the determination of a distance control signal for the invention.

[0130] Fig. 14 shows a diagram of an exemplary modulation signal 140, which is applied in a modulation cycle between times t0 and t1, here as the electrical voltage of an electrically adjustable lens. For simplicity, only one half of a modulation cycle is shown (in the second half, the modulation signal drops again mirror-symmetrically to t1, not shown in detail). The value M of the modulation signal 140 (plotted upwards) changes as a function of time t (plotted to the right) between a minimum Mmin and a maximum M ma x. According to the calibration carried out, if the value M so ii of the modulation signal 140, which at time t S0 n is reached, the focus of the extracted part of the sample light will be at the location of the input aperture of the analysis detector, and accordingly a detector signal 150 of the analysis detector will be maximum. M soii is also called target focus elongation, and t S0 n is also called target focus time.

[0131] At a location on the sample located during scanning, the sample is now to be moved with its local surface to the desired distance from the microscope objective (according to the calibration). For this purpose, the detector signal is measured at the analysis detector over a modulation cycle (or a plurality of modulation cycles, over which an average is calculated), with the distance control signal having a current value ABStoday. The distance control signal can, for example, be a control voltage applied to a linear gantry in the z-direction of the sample's movement device. Typically, there is initially a detuning, i.e., the sample is not at the desired distance from the microscope objective. In Fig.Figure 15 shows an exemplary detector signal 150 measured during a detuning, with the detector signal strength D (plotted upwards) at the analysis detector as a function of time t (plotted to the right) in the modulation cycle between t0 and t1. In fact, due to the detuning present, the maximum of the detector signal 150 occurs at time t. x (instead of t so n) in the modulation cycle. This time t x is also called (current) focus time and corresponds to a value M as shown in Fig. 14 x of the modulation signal 140. M x is also called the (instantaneous) focus elongation. If the applied modulation signal 140 is described by a function M(t), then M(t x ) = M x and M (tsoll) = Msoii .

[0132] The difference M x -M S oii (or alternatively t x -t Soii) indicates the degree of detuning, i.e., how much ABS has deviated from the target value ABSziei required for focusing at the current measurement location on the sample surface for the distance control signal. This difference therefore provides an indication of what change AABS should be applied next to the distance control signal to quickly achieve focusing. The relationship between the difference M x -M S oii (or t x -tsoii) and the change to be made AABS of the distance control signal ABS is strictly monotonic and should be assumed to be linear in this variant (linear control function).

[0133] Then the change AABS to be made can be determined as follows: AABS=(M X -M SO II)*K1, where the constant Kl is a system constant inherent in the confocal microscope that usually does not fluctuate noticeably. Alternatively, AABS can also be determined using AABS = (tx -t S oii)*K2, with constant K2 being a system constant inherent in the confocal microscope and usually not fluctuating noticeably.

[0134] The distance control signal is then adjusted to a new value ABSneu as follows, with

[0135] A BS new = ABS previous + AABS .

[0136] After reaching ABSneu you can again t x or M x as described above. Ideally (with good linearity and correct Kl or K2), the new t x now identical to t S0 n will be or will be the new M x now be identical to Msoii, so that the next change to be made is AABS=0, meaning that focus is achieved directly. Any remaining deviations from t x to t S0 n or M x for the so ii can be minimized iteratively.

[0137] When measuring the sample itself, a distinction can be made between two frequently used measurement modes:

[0138] A first measurement mode, called precision mode, is used for particularly precise measurement of the sample. As soon as t x and t S0 n or M x and M so ii (sufficiently) match, i.e., focus has been achieved, the actual measurement of the sample can begin at the current location located during scanning. Then, a subsequent location on the sample can be located, and focusing and measurement of the sample can be performed in an analogous manner, and so on.

[0139] Note that for an iterative adjustment of the distance control signal to the value ABSziei it is sufficient to calculate the difference M x -M S oii (or t x -t Soii) to determine an AABS with the correct sign (corresponding to a "knowledge of direction", see above) and to choose its value not too large, so that the iterative adjustment converges towards focus.

[0140] A second measurement mode, called quasi-continuous mode, is used for particularly fast sample measurements. In this mode, the sample is continuously moved using the movement device to scan a specified area of ​​the surface and perform a discrete number of sample light measurements. During this continuous movement, the sample light is continuously measured. Due to the continuous movement, the sample light measured between neighboring scanning points contains spatially averaged information about the sample properties. In this quasi-continuous measurement mode, the defocusing of the sample is continuously corrected according to the invention.For this purpose, a closed control loop, for example a PID controller, is used in the electronic control device, which continuously generates a distance control signal in response to the current detuning, thus allowing the movement device to make constant corrections in order to achieve the most precise focus of the sample at any given time.

[0141] In summary, the invention describes a confocal microscope (1) comprising

[0142] - an excitation light source (5) for an excitation light beam (6),

[0143] - an excitation light beam splitter (7),

[0144] - a point detector (14) with detector optics (13) for sample light (10) returning from a sample (2),

[0145] - a microscope lens (4),

[0146] - a movement device (3) for setting a relative distance (AB) between the microscope objective (4) and the sample (2), - an analysis device (16) comprising an analysis beam splitter (8) for coupling out a part (11a) of the sample light (10) returning from the sample (2), an analysis optics (12) for focusing the decoupled part (11a) in the direction of an analysis detector (17), the analysis detector (17) for detecting the decoupled part (11a), and a modulation device (22) with which the refractive power of the analysis optics (12) and / or a relative distance (RA) between the analysis detector (17) and the analysis optics (12) can be cyclically varied in time according to a modulation signal (140), wherein a focus time (t x) is determined at which the decoupled part (11a) is focused on the analysis detector (17), and with which a distance control signal for the movement device (3) is generated. The confocal microscope enables simple and reliable automatic tracking of the distance between the sample and the microscope objective.

[0147] iste:

[0148] 1 confocal microscope

[0149] 2 Sample

[0150] 3 movable sample holder / movement device

[0151] 4 microscope lens

[0152] 4a Exit pupil of the microscope objective

[0153] 5 Excitation light source

[0154] 5a Output aperture of the excitation light source

[0155] 5b Lens of the excitation light source

[0156] 6 Excitation light beam

[0157] 7 Excitation light beam splitters

[0158] 8 analysis beam splitters

[0159] 9 sample-side beam focus

[0160] 10 Sample light 11a Decoupled part of the sample light lib Non-decoupled part of the sample light 11c Remaining part of the sample light lid First part of the sample light

[0161] 12 Analysis optics 12a Entrance pupil of the analysis optics

[0162] 13 Detector optics 14 Point detector

[0163] 14a Entrance aperture of the point detector 14b Spectrograph 15 Point detector-side beam focus 16 Analysis device

[0164] 17 Analysis detector 17a Input panel of the analysis detector

[0165] 18 Relay optics 19 Electrically adjustable lens (ETL)

[0166] 20 non-adjustable lens 21 analysis detector side beam focus

[0167] 22 Modulation device

[0168] 23 electronic control device

[0169] 30 beam deflection wedge

[0170] 41 last lens of the analysis optics

[0171] 42 Focus (when in focus)

[0172] 43 Focus (too far away during test)

[0173] 44 Focus (too close for sample)

[0174] 45 focus (at medium refractive power)

[0175] 46 Focus (with increased refractive power)

[0176] 47 Focus (with reduced refractive power)

[0177] 48 Wobble device

[0178] 50 Focus (slight detuning, sample slightly too far away)

[0179] 51 Focus (larger detuning, sample clearly too far away)

[0180] 90 relay optics

[0181] 101 Entrance Level

[0182] 102 Exit level

[0183] 103 Fourier plane

[0184] 140 modulation signal

[0185] 150 detector signal

[0186] AB (current) distance between sample / local sample surface and microscope objective

[0187] ABsoii Target distance between sample / local sample surface and microscope objective

[0188] ABS distance control signal

[0189] ABSPrevious value of the distance control signal

[0190] ABSnew new value of the distance control signal

[0191] ABSziei Target value of the distance control signal

[0192] AABS Change of the distance control signal

[0193] BFL_i back focal length of element group i

[0194] D Strength / intensity of the detector signal d(l,2) Distance between element groups 1 and 2

[0195] El.l (first) imaging optical element of the first element group El.2 (second) imaging optical element of the first element group E2.1 (first) imaging optical element of the second element group FFL_i Front focal length of element group i

[0196] Gl first mapping element group

[0197] G2 second imaging element group

[0198] GN last mapping element group i Index of element groups j Index of elements in an element group

[0199] M value of the modulation signal

[0200] M(t) function of the modulation signal

[0201] Mmax Maximum value of the modulation signal

[0202] Mmin Minimum value of the modulation signal

[0203] Msoii Target value of the modulation signal / Target focus elongation M x Value of the modulation signal at the maximum of the detector signal / at

[0204] Time t x / current focus elongation

[0205] N Number of element groups

[0206] RA relative distance analysis optics to analysis detector

[0207] SCI section of the beam path of the decoupled part

[0208] SC2 Section of the beam path of the decoupled part

[0209] SW1 beam width (input side)

[0210] SW2 Beam width (output side) t Time tsoii Target time of the maximum of the detector signal / Target focus-

[0211] Time t x Time at maximum of the detector signal / current focus

[0212] Time to Start time of half the modulation cycle ti End time of half the modulation cycle x Direction y Direction z Direction

Claims

Patent claims 1. Confocal microscope (1) for imaging a sample (2), in particular the surface of the sample (2), comprising - an excitation light source (5), in particular an excitation laser, for generating an excitation light beam (6), - an excitation light beam splitter (7) for coupling the excitation light beam (6), - a point detector (14) for registering a sample light (10) caused by the excitation light beam (6) and returning from the sample (2), in particular from the surface of the sample (2), - a microscope objective (4) for focusing the excitation light beam (6) onto the sample (2), in particular onto the surface of the sample (2), and for collecting the sample light (10) returning from the sample (2), - a detector optics (13) for imaging the sample light (10) returning from the sample (2) and collected by the microscope objective (4) onto the point detector (14), - a movement device (3) for adjusting a relative distance (AB) between the microscope objective (4) and the sample (2) according to a distance control signal (ABS), - an analysis device (16) comprising an analysis beam splitter (8) for coupling out a part (11a) of the sample light (10) returning from the sample (2), an analysis optics (12) for focusing only the decoupled part (11a) of the sample light (10) in the direction of an analysis detector (17), and the analysis detector (17) for detecting the decoupled part (11a) of the sample light (10), wherein the analysis Device (16) is designed to generate the distance control signal (ABS) for the movement device (3), further wherein the analysis device (16) comprises a modulation device (22) with which the refractive power of the analysis optics (12) and / or a relative distance (RA) between the analysis detector (17) and the analysis optics (12) can be changed cyclically in time according to a modulation signal (140), wherein the analysis device (16) is designed to determine a focus time (t x ) at which the coupled-out part (11a) of the sample light (10) is focused on the analysis detector (17), and that the analysis device (16) is designed to use the focus time (t x ) to generate the distance control signal (ABS) for the movement device (3) in the respective modulation cycle.

2. Confocal microscope (1) according to claim 1, characterized in that the analysis device (16) is designed to use the focus time (t x ) to indirectly generate the distance control signal (ABS) for the movement device (3) in the respective modulation cycle by an elongation (M x ) of the modulation signal (140) at the focus time (t x ) is read out, and the distance control signal (ABS) for the movement device (3) is generated by means of this elongation (M x ) of the modulation signal (140) at the focus time (t x ) is generated.

3. Confocal microscope (1) according to claim 1 or 2, characterized in that the analysis optics (12) comprises an electrically adjustable lens (19) whose refractive power can be varied cyclically over time by means of the modulation signal (140).

4. Confocal microscope (1) according to claim 3, characterized in that a frequency of the modulation signal (140) corresponds to a natural frequency of the electrically adjustable lens (19).

5. Confocal microscope (1) according to claim 3 or 4, characterized in that the analysis optics (12) comprises the electrically adjustable lens (19) on the input side and further comprises a non-adjustable, imaging optical element, in particular a non-adjustable lens (20), on the output side.

6. Confocal microscope (1) according to one of claims 3 to 5, characterized in that the electrically adjustable lens (19) is aligned horizontally.

7. Confocal microscope (1) according to claim 1 or 2, characterized in that the analysis detector (17) is arranged on a wobble device (48) with which the position of the analysis detector (17) in the propagation direction (x) of the decoupled part (11a) of the sample light (10) can be changed cyclically over time, in particular wherein the analysis optics (12) are designed to be stationary.

8. Confocal microscope (1) according to one of the preceding claims, characterized in that a relay optics (18; 90) is arranged between the analysis beam splitter (8) and the analysis optics (12), with which the exit pupil (4a) of the microscope objective (4) is imaged onto an entrance pupil (12a) of the analysis optics (12) with an imaging factor ABF.

9. Confocal microscope (1) according to claim 8, characterized in that the relay optics (18; 90) comprises an even number N of imaging element groups (Gi), each imaging element group (Gi) having at least one imaging comprising an optical element (E1.1, E1.2, E2.1), wherein each imaging element group i (Gi) can be assigned a front focal length FFL_i, a rear focal length BFL_i and an effective focal length EFL_i, with i: index of the imaging element groups (Gi), and that at least approximately the exit pupil (4a) of the microscope objective (4) is positioned at a distance FFL_1 in front of the first imaging element group i = l (G1), and the entrance pupil (12a) of the analysis optics (12) is positioned at least approximately at a distance BFL_N behind the last imaging element group i = N (G1), and adjacent element groups i, i + 1 (Gi, Gi + 1) are positioned at least approximately at a distance d(i,i + 1) = BFL_i + FFL_i + 1 from one another.

10. Confocal microscope (1) according to claim 9, characterized in that at least one imaging element group (Gi) comprises at least two imaging optical elements (El.1, El.2, E2.1).

11. Confocal microscope (1) according to claim 9 or 10, characterized in that the first imaging element group (Gl) comprises at least two imaging optical elements (El.l, El.2), and these imaging optical elements (El.l, El.2) are selected such that FFL_1>BFL_1.

12. Confocal microscope (1) according to one of claims 8 to 11, characterized in that the imaging factor ABF=1.

13. Confocal microscope (1) according to one of claims 8 to 11, characterized in that the imaging factor ABF is magnifying, with ABF > 1, in particular where 1 < ABF < 3.

14. Confocal microscope (1) according to one of the preceding claims, characterized in that the analysis detector (17) has an entrance aperture (17a) with a diameter which is not greater than the diameter of the part (11a) of the sample light (10) which is completely focused by means of the analysis optics (12), and in that the analysis device (16) is designed to determine the focus time (t x ) by measuring the intensity (D) of the detector signal (150) in the modulation cycle at the focus time (t x ) is maximum.

15. Use of a confocal microscope (1) according to one of the preceding claims for imaging a sample (2), in particular a surface of the sample (2), by means of confocal microscopy, in particular confocal light microscopy, confocal Raman microscopy or confocal fluorescence microscopy, wherein the sample (2) is measured successively at a plurality of measuring locations, wherein for a change of the measuring location the sample (2) is moved transversely to the propagation direction (z) of the excitation light beam (6), and wherein with the distance control signal (ABS) generated by the analysis device (16) the relative distance (AB) between the microscope objective (4) and a local surface of the sample (2) at the respective measuring location is kept at least approximately constant across the various measuring locations with the movement device (3).

16. Method for imaging a sample (2), in particular the surface of the sample (2), with confocal microscopy, in particular with confocal light microscopy, confocal Raman microscopy or confocal fluorescence microscopy, and in particular wherein the method runs on a confocal microscope (1) according to one of claims 1 to 14, comprising the following steps: - An excitation light beam (6) is generated by an excitation light source (5), in particular an excitation laser, - The excitation light beam (6) is coupled in using an excitation light beam splitter (7); - A point detector (14) registers a sample light (10) caused by the excitation laser beam (6) and returning from the sample (2), in particular from the surface of the sample (2), - Using a microscope objective (4), the excitation light beam (6) is focused onto the sample (2), in particular onto the surface of the sample (2), and the sample light (10) returning from the sample (2) is collected, - With a detector optics (13), the collected sample light (10) returning from the sample (2) is imaged onto the point detector (14); - A movement device (3) is used to set a relative distance (AB) between the microscope objective (4) and the sample (2) according to a distance control signal (ABS), - The distance control signal (ABS) for the movement device (3) is generated by an analysis device (16) comprising an analysis beam splitter (8) which couples out a part (11a) of the sample light (10) returning from the sample (2), an analysis optics (12) which focuses only the coupled-out part (11a) of the sample light (10) in the direction of an analysis detector (17), and the analysis detector (17) which detects the coupled-out part (11a) of the sample light (10), further wherein the analysis device (16) comprises a modulation device (22) with which the refractive power of the analysis optics (12) and / or a relative distance (RA) between the analysis detector (17) and the analysis optics (12) is changed cyclically over time according to a modulation signal (140), wherein the analysis device (16) in a respective modulation cycle of the modulation signal (140) based on a detector signal (150) of the analysis detector (17) a focus time (tx ) at which the coupled-out part (11a) of the sample light (10) is focused on the analysis detector (17), and wherein the analysis device (16) uses the focus time (t x ) generates the distance control signal (ABS) for the movement device (3) in the respective modulation cycle.

17. Method according to claim 16, characterized in that the point detector (14) is designed as a spectrograph (14b) and a spectral composition of the light coming back from the sample (2) Light (10) is analyzed with the spectrograph (14b), and that in the sample light (10) returning from the sample (2) fluorescence properties and / or Raman scattering properties of the sample (2) are determined, in particular wherein in front of the point detector (14) with a filter of the Sample (2) reflected sample light (10), which has the same wavelength as the excitation light beam (6), is blocked.

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